We have built a laser based optical light scattering sensor to directly detect and identify bacterial colonies as they grow on agar surface in Petri-dish. Upon shining of laser on the bacterial colony, it generates unique scatter signature for each colony that is captured by a CCD camera. Image analysis software allows identification and classification of bacteria at genus, species and even at serovar level in minutes. Selective and chromogenic agar media help generate unique differential scatter signatures for bacteria and it is highly reproducible and specific. BARDOT has been extensively studied for foodborne bacterial identification including Listeria, Salmonella, Escherichia coli, Vibrio, Bacillus and other pathogens. BARDOT is licensed to Advanced Bioimaging Systems, LLC (West Lafayette, IN).

Cell-Based Sensor (CBS):

Pathogens inherently interact with mammalian cells during infection. Pathogens also display specific cell or tissue tropism. Use of appropriate mammalian cells of different tissue origin such as those from blood, liver, kidney, intestine, skin, etc can allow detection of pathogens or toxins very quickly. Our laboratory is developing cell-based sensor using various cell lines in array format in 96-well plate to rapidly asses the presence or absence of pathogens or toxins in food or beverage samples for potential application in food safety and food defense. In CBS, mammalian cells, engineered or natural are grown in three-dimensional configuration using hydrogel-collagen matrix and pathogen-induced cellular damage is monitored optically or electrically. Certain microbial toxins can be detected at nanogram quantities in 15-20 min while pathogenic bacterial cells are detected in 2-3 h. This system is ideal for detection of viable harmful bacteria and active toxins.

Microfluidic Biochip:

Lab-on-a chip platform captures bacteria, monitors growth and then detect. Dielectrophoresis (DEP) as part of the chip set up, helps concentrate bacterial cells on the silicon dioxide chip surface, pre-coated with pathogen specific antibodies or bio-receptor molecules for specificity. Low conductive growth media flown through the chip promote bacterial growth and impedance analyzer monitors resulting changes in the conductivity of the media. On chip PCR assay is now being developed to detect captured bacteria. Detection of target pathogens at low levels is possible in less than 6 h. Our contribution to this multidisciplinary project is to generate specific antibodies or bio-receptor molecules, media development, assay validation and microbiological analysis. Biochip platform has been optimized for L. monocytogenes detection. Our current effort is on Salmonella and Shiga-toxigenic E. coli.

Fiber optic biosensor:

Fundamental property of optical fiber is its ability to deliver the incoming light signal, and to act as a transducer of outgoing light. In fiber optic immunosensor, antibodies or bioreceptors in sandwich configuration detect pathogens and toxins. Captured target analyte on fiber optic waveguide is detected by using a fluorophore labeled antibody/bio-receptor. As laser is shined on the top of the fiber, it excites the fluorophore, generating evanescent wave, which returns to the detector. Signal is proportional to the amount of analyte bound to the fiber surface. The detection limit for three major foodborne pathogens, Salmonella, E. coliO157:H7 and L. monocytogenes is about 103 cells/ml. We have also demonstrated that all three pathogens can be detected simultaneously if present in the same sample.

Surface Plasmon Resonance (SPR) biosensor:

We are using SPR to study protein-protein interaction i.e., between Hsp60 and Listeria Adhesion Protein (LAP) in L. monocytogenes and between intimin and translocated intimin receptor (TIR) in E. coli O157:H7.

·Influence of environmental or food–related stress on bacterial capture and detection

·Cloning and characterization of bio-receptoras capture molecule for foodborne pathogens

·Bacterial growth media development for biosensor-based detection

·Sample processing and preparation strategies using affinity based separation methods such as paramagnetic beads and ion exchange resins in a pathogen enrichment device (PED)

Understanding the molecular and cellular mechanism of intracellular Listeria monocytogenes colonization and translocation through epithelial barrier during intestinal phase of infection

Adhesion of L. monocytogenes to intestinal cells is an important initial event in Listeria pathogenesis. Our research group has discovered a 104-kDa Listeria adhesion protein, designated LAP that plays an important role during the intestinal phase of L. monocytogenes infection. LAP is present in all Listeria spp. including two newly reported listeriae; L. marthii and L. rocourtiae (unpublished) but absent in L. grayi. It functions as a housekeeping enzyme, an alcohol acetaldehyde dehydrogenase (AAD; lmo 1634) consisting of 866 amino acids with an N-terminal acetaldehyde dehydrogenase (ALDH), and a C-terminal alcohol dehydrogenase (ADH). A putative NAD+ binding domain is located between Gly427–Gly432, and an Fe2+ binding domain is located between Gly724–Gly742. Although it is present in both pathogenic and nonpathogenic Listeria species, only pathogenic bacteria secrete and re-associate LAP onto the cell surface to promote host cell interaction. The host cell receptor is a mitochondrial chaperonin called heat shock protein 60 (Hsp60). The N-terminal Gly224-Gly411 in ALDH domain interact with Hsp60. We showed that LAP–Hsp60 interaction promotes transepithelial translocation of L. monocytogenes through a paracellular route, suggesting an alternate strategy for bacteria to cross epithelial barriers during the intestinal phase of infection. Furthermore, L. monocytogenes infection at low dosage also increases Hsp60 expression, promoting enhanced LAP-mediated bacterial translocation.